DNA damage-dependent cyclin D1 proteolysis: GSK3beta holds the smoking gun.

Ubiquitin mediated degradation of cyclin D1 following the G(1)/S transition counters its mitogen-dependent accumulation during G(1) phase of the cell cycle. Although the cellular machinery responsible for this process has been identified, how this regulatory pathway interfaces with cellular stress responses, often referred to as checkpoints, remains to be established. One intensely investigated checkpoint is the cellular response to DNA damage. When DNA damage is sensed, the corresponding DNA damage checkpoint triggers the inhibition of CDK-dependent cell cycle progression, with arrest coordinated by induction of CDK inhibitors and rapid degradation of specific cyclins, such as cyclin D1. In recent work, we identified a phosphorylation- and Fbx4-dependent cyclin D1 degradation mechanism in response to genotoxic stress.(18) This work revealed that loss of cyclin D1 regulation compromises the intra-S-phase response to DNA damage, promoting genomic instability and sensitization of cells to S-phase chemotherapy, highlighting a potential therapeutic strategy for cancers exhibiting cyclin D1 accumulation.

Genotoxic stress-induced cyclin D1 phosphorylation and proteolysis are required for genomic stability.

While mitogenic induction of cyclin D1 contributes to cell cycle progression, ubiquitin-mediated proteolysis buffers this accumulation and prevents aberrant proliferation. Because the failure to degrade cyclin D1 during S-phase triggers DNA rereplication, we have investigated cellular regulation of cyclin D1 following genotoxic stress. These data reveal that expression of cyclin D1 alleles refractory to phosphorylation- and ubiquitin-mediated degradation increase the frequency of chromatid breaks following DNA damage. Double-strand break-dependent cyclin D1 degradation requires ATM and GSK3beta, which in turn mediate cyclin D1 phosphorylation. Phosphorylated cyclin D1 is targeted for proteasomal degradation after ubiquitylation by SCF(Fbx4-alphaBcrystallin). Loss of Fbx4-dependent degradation triggers radio-resistant DNA synthesis, thereby sensitizing cells to S-phase-specific chemotherapeutic intervention. These data suggest that failure to degrade cyclin D1 compromises the intra-S-phase checkpoint and suggest that cyclin D1 degradation is a vital cellular response necessary to prevent genomic instability following genotoxic insult.

Speeding through cell cycle roadblocks: Nuclear cyclin D1-dependent kinase and neoplastic transformation.

Mitogenic induction of cyclin D1, the allosteric regulator of CDK4/6, is a key regulatory event contributing to G1 phase progression. Following the G1/S transition, cyclin D1 activation is antagonized by GSK3beta-dependent threonine-286 (Thr-286) phosphorylation, triggering nuclear export and subsequent cytoplasmic degradation mediated by the SCFFbx4-alphaBcrystallin E3 ubiquitin ligase. Although cyclin D1 overexpression occurs in numerous malignancies, overexpression of cyclin D1 alone is insufficient to drive transformation. In contrast, cyclin D1 mutants refractory to phosphorylation-dependent nuclear export and degradation are acutely transforming. This raises the question of whether overexpression of cyclin D1 is a significant contributor to tumorigenesis or an effect of neoplastic transformation. Significantly, recent work strongly supports a model wherein nuclear accumulation of cyclin D1-dependent kinase during S-phase is a critical event with regard to transformation. The identification of mutations within SCFFbx4-alphaBcrystallin ligase in primary tumors provides mechanistic insight into cyclin D1 accumulation in human cancer. Furthermore, analysis of mouse models expressing cyclin D1 mutants refractory to degradation indicate that nuclear cyclin D1/CDK4 kinase triggers DNA re-replication and genomic instability. Collectively, these new findings provide a mechanism whereby aberrations in post-translational regulation of cyclin D1 establish a cellular environment conducive to mutations that favor neoplastic growth.

Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication.

Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.

Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss.

Developmental abnormalities, cancer, and premature aging each have been linked to defects in the DNA damage response (DDR). Mutations in the ATR checkpoint regulator cause developmental defects in mice (pregastrulation lethality) and humans (Seckel syndrome). Here we show that eliminating ATR in adult mice leads to defects in tissue homeostasis and the rapid appearance of age-related phenotypes, such as hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. Histological and genetic analyses indicate that ATR deletion causes acute cellular loss in tissues in which continuous cell proliferation is required for maintenance. Importantly, thymic involution, alopecia, and hair graying in ATR knockout mice were associated with dramatic reductions in tissue-specific stem and progenitor cells and exhaustion of tissue renewal and homeostatic capacity. In aggregate, these studies suggest that reduced regenerative capacity in adults via deletion of a developmentally essential DDR gene is sufficient to cause the premature appearance of age-related phenotypes.